Project Summary Bone is a dynamic organ, undergoing constant remodeling by osteoblasts that make new bone, and osteoclasts that degrade bone. The resorption of bone is the unique function of the osteoclast, a cell type derived from hematopoietic cells in the myeloid lineage. Osteoclast development requires receptor stimulation through RANK (receptor for activation of NFkappaB), and c-fms (receptor for macrophage colony stimulating factor, M-CSF). Recently, we and others demonstrated an additional requirement for costimulatory signals through ITAM (immunoreceptor tyrosine based activation motif) signaling receptors during normal osteoclast differentiation and function. Interestingly we also found that ITAM-adapter deficient mice lose bone under conditions of rapid bone remodeling induced by estrogen deficiency, a low calcium diet or LPS (lipopolysaccharide). Thus, other costimulatory signals and/or receptors can substitute for ITAM-adapter mediated signals in osteoclastogenesis under these pathologic conditions of bone remodeling. In osteoclastogenesis, ITAM-signaling receptors have been thought to primarily provide a Ca2+ signal to activate the critical transcription factor for osteoclastogenesis, NFATc1 during RANKL stimulation. We are interested in determining what other signals are provided by ITAM-signaling receptors that are also critical for osteoclastogenesis to help us understand the requirements for costimulatory signals. In other cell types, ITAM- signals also lead to production of reactive oxygen species (ROS). ROS have been previously demonstrated to be important regulators of osteoclast differentiation and function in vitro and in vivo. RANKL has been shown to stimulate ROS production during osteoclastogenesis. We observed that RANKL stimulated ROS production in preosteoclasts does not occur in the absence of ITAM adapters. Our proposal will investigate the hypothesis that ITAM-adapters provide both a Ca2+ signal and a ROS signal to osteoclast precursors that together are required for osteoclastogenesis. We propose that without ITAM- signaling in osteoclast precursors, osteoclasts can only be generated under stressful or inflammatory conditions where reactive oxygen species are prevalent. Our hypothesis suggests that osteoclasts differentiate under distinct stimuli under basal and inflammatory conditions, and may reveal subtypes of osteoclasts that can be differentially regulated. We propose the following specific aims: 1. Determine the effect of ROS stimulation on the ITAM-signaling pathway and the mechanism of enhanced osteoclastogenesis by ROS. 2. Determine the requirement for ITAM-adapters and ITAM adapter signals for production of reactive oxygen species (ROS) during RANKL stimulated osteoclastogenesis. 3. Determine the requirement for ROS during rapid osteoclastogenesis in the absence of ITAM- adapter signals. PUBLIC HEALTH RELEVANCE: Project Narrative Osteoclasts function to resorb bone throughout life. Many diseases including osteoporosis, rheumatoid arthritis, and cancer metastases involve significant bone loss due to inappropriate osteoclast activation. The incidence of osteoporosis increases with aging in men and women and is common in the VA patient population. Treatment of osteoporotic fractures is costly with greatly increased morbidity and mortality in patients with an osteoporotic vertebral or hip fracture. We are interested in determining how the signals required for osteoclasts to develop under stressful or inflammatory conditions differ from the signals that develop osteoclasts under basal conditions. Our studies are important to facilitate development of novel therapeutics to prevent early bone loss and fracture in our patients. Our proposal directly applies to the designated priority areas of research including aging, chronic disease, cancer and degenerative joint disease.